1. Field of the Invention
The present invention relates generally to the cyclooxygenases and their roles in human pathology, including cancer and inflammation. More particularly, this invention pertains to methods and articles of manufacture for detecting or measuring COX-2 activity by detecting and measuring COX-2 specific enzymatic products.
2. Description of the Related Art
COX is a prostaglandin endoperoxide synthase enzyme (cyclooxygenase, COX, EC 1.14.99.1), which catalyzes the conversion of arachidonic acid to prostaglandin (PG) H2. Two isoforms of COX are known, COX-1 and COX-2. COX-1 is constitutively expressed. COX-2, however, is inducible in a variety of cells, especially those of the central nervous and immune systems (Masferrer et al. 1994, Proc. Natl. Acad. Sci. USA 91:3228-3232; Vane et al. 1994, Proc. Natl. Acad. Sci. USA 91:2046-2050; Kennedy et al. 1993, Biochem. Biophys. Res. Commun. 197:494-500). Certain changes in COX-2 activity are associated with a variety of human inflammatory diseases. These diseases include, but are not limited to, acute appendicitis, asthma, myocardial infarction, certain immunological disease processes, infection, malignancy, endotoxemia and reperfusion injury. In addition, COX-2 inappropriate expression or overexpression is associated with certain types of cancers, including, but not limited to, carcinoma of the colon, rectum, stomach, esophagus, lung, and skin. The amount of COX-2 expression is related to the stage or progression of cancer (Fosslien, E, et al. 2000, Ann. Clin. Lab. Sci. 30:3-21). COX-2 has become a major pharmaceutical target for developing treatments for these and other diseases.
Nonsteroidal anti-inflammatory drugs prevent hyperalgesia and inflammation by inhibiting the cyclooxygenase-2 (COX-2) catalyzed oxygenation of arachidonic acid to prostaglandin (PG) H2. The lipoamino acid N-arachidonylglycine (NAGly), has also been shown to suppress tonic inflammatory pain, and is naturally present at significant levels in many of the same mammalian tissues that express COX-2. The present inventors have discovered that COX-2 selectively metabolizes NAGly to PGH2 glycine (PGH2-Gly) and hydroxyeicosatetraenoic glycine (HETE-Gly). Site-directed mutagenesis experiments identify the side pocket residues of COX-2, especially Arg-513, as critical determinants of the COX-2 selectivity towards NAGly. Additionally, the present inventors have discovered that NAGly is a charged arachidonyl derivative that is a selective substrate for COX-2, allowing for easier detection in some instances. Accordingly, the present inventors have discovered the role for COX-2 in the regulation of lipoamino acid levels, including NAGly levels, and the formation of a novel class of eicosanoids from NAGly metabolism.
Yu et al. (1997) J. Biol. Chem. 272:21181-21186, describes the enzymatic conversion of arachidonyl ethanolamide (anandamide, AEA), to PGE2-ethanolamide in cell lines expressing COX-2 but not COX-1.
U.S. Pat. No. 5,543,297 to Cromlish et al., describes measuring total COX activity (COX-1 activity and COX-2 activity) in separate samples, with and without a COX-2 specific inhibitor, and then indirectly estimating COX-2 specific activity by subtracting the total COX activity observed with the inhibitor from the total COX activity observed without the inhibitor. One major weakness of this method is that the dynamics of enzymatic inhibition change based upon numerous variables including time, temperature, concentration, specificity, sample preparation, etc.
U.S. Pat. No. 5,475,021 to Marnett et al. describes a method of measuring the activity of purified COX-2 by measuring O2-uptake during catalysis. This method requires purification of the enzyme.
U.S. Pat. No. 6,045,773 to Isakson et al., describes a method for measuring COX-2 activity in a mammal by administering a positron-emitting radioisotope-labeled COX-2 selective binding agent to the mammal and then detecting the label by positron-emission tomography (PET). Weaknesses of this method include the invasive nature and expense of PET equipment. In addition, the method only localizes COX-2 protein but does not detect or measure activity.
Methods of detecting and measuring COX-2 activity are highly desired. What is needed, then, is a less-invasive, direct method of selectively detecting and measuring COX-2 activity in biological samples and whole animals without the need to purify the enzyme.